Microarray line scanning method and system

ABSTRACT

A line scanning arrangement for imaging microarrays includes a line illuminator that converts output from one or more lasers to a radiation line. The laser output passes through a single mode fiber and a module that converts the laser light to the radiation line. The line is confocally directed to sites on the microarray, and retrobeams returned from the sites are collected on an imaging detector. The microarray is moved in the imaging apparatus so as to progressively illuminate an array or matrix of sites for imaging.

BACKGROUND

The present invention relates generally to the field of scanners forimaging and evaluating biological microarrays. More particularly, theinvention relates to a technique for rapidly and accurately evaluatingmicroarrays through the use of confocal line scanning.

An increasing number of applications are being developed for biologicalmicroarrays. Such microarrays typically include Deoxyribonucleic Acid(DNA) and Ribonucleic Acid (RNA) probes that are specific for nucleotidesequences present in genes in humans and other organisms. Individual DNAor RNA probes can be attached at specific locations in a small geometricgrid on a microarray support. A test sample, such as from a known personor organism, can be exposed to the grid, such that complimentary genesor fragments hybridize to probes at the individual sites in the array.The array can then be examined by scanning specific frequencies of lightover the sites to identify which genes or fragments in the sample werepresent, by fluorescence of the sites at which genes or fragmentshybridized.

Such microarrays, sometimes referred to as gene or genome chips, DNAchips, gene arrays, and so forth, may be used for expression profiling,monitoring expression levels, genotyping, sequencing, and so forth. Forexample, diagnostic uses may include evaluation of a particularpatient's genetic makeup to determine whether a disease state is presentor whether pre-disposition for a particular condition exists. Thereading and evaluation of microarrays is a key to their utility. Forexample, in certain types of microarrays, DNA probes are attached tobeads at individual sites in the array. Because the fragments areattached in a random or statistically varying pattern, it is necessaryto image the microarray to determine the location of each of theindividual fragments, and their makeup. Moreover, once a sample has beenexposed to the microarray, reading the microarray is necessary todetermine the makeup of the sample.

Various types of microarray readers have been proposed and are currentlyin use. In many such readers, a small point of light is scanned acrosslines of the microarray to cause fluorescence of the individual sites,particularly those sites to which genes or fragments are hybridized.Such scanning is preferably extremely fast and accurate. Currentmicroarray designs, provide for many thousands of individual sites in avery small area of the substrate. The number of sites and the density ofsuch sites in the array are constantly increasing, posing challenges toknown scanning and imaging techniques.

There is a constant need, therefore, for improved microarray scanningand imaging technologies. There is a particular need for a techniquethat will allow for very fast scanning of a large number of individualsites, and that reduces the potential for errors in imaging.

BRIEF DESCRIPTION

The present invention provides a microarray scanning and imaging systemdesigned to respond to such needs. In accordance with certain aspects ofthe invention, a laser light source is coupled to a single mode fiberoptic cable for transmitting laser light in single mode transmission. Aline illuminator converts laser light from the source to a radiationline. The line illuminator includes a collimator arranged to receive thelaser light from the source and an aspherical lens for convertingcollimated light from the collimator to the line of radiation. Afocusing device directs the line of radiation onto a plane at thesurface of a microarray.

In accordance with another aspect of the invention, a similar system mayinclude a second fiber optic cable coupled to the first single modefiber optic cable at one end thereof and to the line illuminator atanother end thereof, at least one of the fiber optic cables being asingle mode fiber optic cable.

In accordance with yet another aspect of the invention, a system forimaging a microarray includes first and second laser light sources, eachsource configured to output laser light in a different predeterminedfrequency band. First and second single mode fiber optic cables arecoupled to the first and second laser light sources, respectively, fortransmitting laser light in single mode transmission. First and secondline illuminators are coupled to the first and second single mode fiberoptic cables, respectively, for converting laser light from therespective source to a line of radiation, the line illuminators eachincluding a collimator arranged to receive the laser light from thesource and an aspherical lens for converting collimated light from thecollimator to the line of radiation. A focusing device, then directs theline of radiation onto a plane at the surface of a microarray.

The invention also provides methods for making and using arrangements ofthe type discussed above, and more particularly described in thediscussion provided below.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 is a diagrammatical overview of a microarray scanning system forconfocal line scanning of a microarray in accordance with aspects of thepresent technique;

FIG. 2 is a diagrammatical perspective view of a portion of a microarrayillustrating an exemplary manner in which a radiation line is directedtoward regions of the microarray in which sites are located that are tobe imaged;

FIG. 3 is a more detailed diagrammatical representation of a portion ofa microarray that is illuminated by a confocal radiation line to imagethe sites on the microarray in accordance with the present technique;

FIG. 4 is a diagrammatical perspective view of a combined radiation linedirected toward a surface of a microarray to confocally irradiate siteson the array, and to confocally return radiation to a detector inaccordance with aspects of the present technique;

FIG. 5 is a similar diagrammatical perspective view illustrating aseries of confocally directed beams of radiation along a line forsimilarly irradiating sites of a microarray in accordance with thepresent technique;

FIG. 6 is a diagrammatical side view of a technique for convertingoutput of a laser to a radiation line for confocal line scanning of amicroarray;

FIG. 7 is a similar, top view of the conversion of the output of a laserto a radiation line for use in the present confocal line scanningtechnique;

FIG. 8 is a graphical representation of an intensity profile for aradiation line produced by the arrangements of FIGS. 6 and 7;

FIG. 9 is a diagrammatical representation of a first exemplaryconfiguration for a modular arrangement used in converting output of alaser to a radiation line for confocal line scanning in accordance withthe invention;

FIG. 10 is an alternative arrangement for conversion of a laser outputto a radiation line in accordance with the present invention;

FIG. 11 is a further alternative arrangement for converting laser outputto a radiation line in accordance with the invention;

FIG. 12 is yet another alternative configuration for converting laseroutput to align a radiation;

FIG. 13 is a sectional view of an exemplary line generator modulesuitable for use in accordance with the invention;

FIG. 14 is a diagrammatical overview for a scanning system that includestwo laser beams, the output of which is combined for confocal linescanning of a microarray;

FIG. 15 is a diagrammatical overview of an alternative arrangement formulti-wavelength confocal line scanning of a microarray;

FIG. 16 is an opto-mechanical diagrammatical representation of apresently contemplated implementation for multi-wavelength confocal linescanning of a microarray;

FIG. 17 is a diagrammatical view of a series of individual sites on abiological microarray, illustrating how the confocal line scanning ofthe present invention improves accuracy by reducing the potentialcrosstalk, particularly in certain types of layout of the sites on themicorarray with respect to radiation lines used in imaging;

FIGS. 18 through 21 are diagrammatical views of exemplary radiation linegenerators that may be suitable for use in the invention;

FIGS. 22 and 23 are diagrammatical views of line generators in afluorescence imaging system, suitable for use in the invention;

FIGS. 24( a)-(c) are diagrams showing the projection of a laser spot ona line scan camera and binning and TDI implementations in accordancewith certain aspects of the invention;

FIG. 25 is a diagrammatical view of an image scanning system that isconfigured to conduct multi-spectral fluorescence imaging in accordancewith aspects of the invention;

FIG. 26 is a block diagram of an exemplary line-scan imaging sensor foruse with the system shown in FIG. 25;

FIG. 27 is a diagrammatical view of a further image scanning system thatis configured to conduct multi-spectral fluorescence imaging;

FIG. 28 is a block diagram of an exemplary line-scan imaging sensor foruse with the system shown in FIG. 27;

FIG. 29 is a block diagram of an exemplary line-scan imaging detectorfor use with the invention; and

FIGS. 30( a)-(b) are block diagrams of other exemplary line-scan imagingdetectors for use with the invention.

DETAILED DESCRIPTION

The present invention provides an image scanning system and architecturehaving rapid scan times while maintaining high resolution and imagequality. These and other advantages result from configuring a detectorarray to achieve confocality in the scanning axis by restricting thescan-axis dimension of the detector array. As set forth in furtherdetail below, an apparatus of the invention can be configured to achieveconfocality in a single axis of a detector array such that confocalityonly occurs in that dimension.

The detector array can have rectangular dimensions such that the shorterdimension of the detector is in the scan-axis dimension. Imaging opticscan be placed to direct a rectangular image of a sample region to thedetector array such that the shorter dimension of the image is also inthe scan-axis dimension. In this way, the detector array forms a virtualslit. A virtual slit configuration provides several advantages over theuse of a typical slit placed in front of a detector. For example,configuring a detector array as a virtual slit reduces the number ofunused array elements compared to a configuration in which a detectorarray, having standard dimensions, is used with a slit. Reducing thenumber of unused elements increases efficiency of data acquisition andreduces image processing time. Furthermore, using a virtual slit allowsboth the detector and slit to be at the focal plane of the projectionlens eliminating any focus compromise of either position or therequirement for a relay lens between the slit and detector.

A detector array configured to have a virtual slit is particularlyuseful when employed in an imaging apparatus that is further configuredto direct a radiation line to a sample. The radiation line can haverectangular dimensions in which the shorter dimension is short enough toachieve confocality in a single axis corresponding to the shorterdimension of the detector array. Thus, confocality can be achieved for,excitation, detection or both. An instrument can be configured to limitexcitation error in the confocal axis such that predominantly all of theexcitation radiation is contained within a spot comparable with theresolution of the instrument.

An apparatus that includes a detector array forming a virtual slit canbe configured to obtain an image of the sample at high resolution, forexample, in the low micron to submicron range. In particularembodiments, an image can be obtained at a Rayleigh resolution between0.2 and 10 micrometers. Furthermore, the ratio of the shorter of the tworectangular dimensions for the rectangular detector array and theproduct of the Rayleigh resolution of the imaging optics multiplied bythe magnification of the imaging optics can be used to determine thesize and dimensions of the virtual slit for achieving confocality in asingle axis. If desired, the ratio of the shorter of two rectangulardimensions for a radiation line to the Rayleigh resolution of theimaging optics can be selected to achieve confocality in a single axis.

Accordingly, an imaging apparatus of the invention can be configured tohave resolution along the length of the line perpendicular to the scanaxis that is matched to the system resolution. For example in a CCDdevice, 4000 CCD elements can be used along the length of a 2 mmradiation line (the horizontal axis) resulting in a 0.5 μm pixelresolution at a sample. The number of CCD elements “n” in the directionperpendicular to the radiation line (the vertical axis) can be chosen tocollect substantially all of the emitted radiation while reducing theamount of unwanted background radiation collected.

An imaging apparatus of the invention can be further configured suchthat all pixel elements in the vertical axis are collected in a common“bin” and read out as a single value. Advantages of the binning approachcompared to a typical Time Delay Integration (TDI) design are that thereadout rate can be reduced by a factor of “n”, the system hasconfocality in one axis, and the tolerance of the synchronization timingof the readout with the y-stage movement can be reduced. It will beunderstood that a TDI design can be configured to have a virtual slit bylimiting the number of vertical pixels. An additional advantage oversystem designs where n=1 are that the collection efficiency of thesystem can be increased and the sensitivity to small optical alignmentdrifts can be decreased.

Turning now to the drawings, and referring first to FIG. 1, an imagingsystem 10 is illustrated diagrammatically as including a scanner 12 inwhich a sample or microarray 14 may be inserted for imaging purposes. Asdescribed more fully below, the microarray 14 includes a substrate orsupport on which an array of sites is formed. Each site including anattached molecular fragment, such as a gene or gene fragment, which mayhave attached thereto a molecule, which may be a complementary moleculein the case of DNA or RNA probes, from a specific sample. In presentembodiments, many thousands of such sites may be provided in rows or agrid pattern in portions or segments on the microarray. The microarrayitself may be formed by various technologies, including, as in a presentembodiment, microbeads. Other microarrays which may be imaged inaccordance with the present techniques may include microarrays formed byphotolithography, and other processes known or developed in the art.

The scanner 12 will include optics described in greater detail below forconfocal line scanning of the sites on microarray 14. In the illustratedembodiment, the scanner is a table-top device having a sample tray 16 inwhich the microarray, or a plurality of microarrays may be positioned.The tray may be configured to advance the microarray 14 into a scanningposition, and subsequently slowly move the microarray, as describedbelow, to allow successive lines on the microarray to be irradiated, andreturn radiation or retrobeams caused by fluorescence of individualsites. The retrobeams are focused on a detector for imaging andanalyzing the sites, also described below. In particular embodiments,multiple retrobeams can be focused to multiple different detectors. Forexample, a retrobeam of a first wavelength can be focused to a firstdetector and a retrobeam of a second retrobeam can be focused to asecond detector, as set forth in further detail below.

Control signals for operation of the scanner 12 originate from acontroller or workstation 18. The workstation 18 also includes softwarefor receiving the imaging signals from the scanner 12. The imagingsoftware of workstation 18 will typically be embodied in a generalpurpose or application-specific computer 20 which also controls andreceives signals from interface components 22, which will typicallyinclude a monitor 24 and input devices 26. The imaging software operablein workstation 18 will preferably provide an intuitive interface forloading and initializing the scanner, for performing imaging scans onmicroarrays, and for saving the data. During the scanning process, thesystem 10 creates individual files for different wavelengths ofradiation used to image the microarray, which may be referred to hereinas red and green channels. These may be provided in a consolidated file.Data and associated images may then be saved in a convenient format,such as a conventional TIFF format, or any other suitable image dataformat or protocol. The workstation 18 may be coupled to other networkcomponents, including down-stream processing and application-specificsoftware for higher-level and data analysis, such as via a networkindicated generally by reference numeral 28 in FIG. 1.

As noted above, the microarray 14 will include a plurality of sitesarranged in portions or regions of a substrate, for example, asindicated generally in FIG. 2. As shown in FIG. 2, the microarray 14 mayinclude a support or substrate 30, which may be a glass, a plastic, asemiconductor, or any other convenient support such as those describedelsewhere herein. On this support 30, one or more sample areas 32 areprovided in which individual sites will be formed, each typicallyprovided with a respective probe molecule used to test a sample. In apresent invention, the sample area 32 is scanned for imaging purposes bya radiation line, indicated generally by reference numeral 34 in FIG. 2.The radiation line is formed by excitation radiation which is confocallydirected along the line 34 to irradiate a plurality of sitessimultaneously, as indicated generally by arrows 36 in FIG. 2. Theindividual sites at which target molecules (e.g., genetic fragments)will have bound are thereby caused to fluoresce due to the presence ofdyes indicative of an interaction of a target with the site, returningradiation as indicated by lines 38 in FIG. 2. As described below, thisreturned radiation, or retrobeam, will be confocally directed toward animaging detector where an image will be made of the line for furtherprocessing and analysis. To permit the sites to be successively imaged,then, the entire microarray may be displaced slowly as indicatedgenerally by reference numeral 40. The line 34 along which the sites areirradiated will thereby generally progress along successive parallellocations on the microarray as the microarray is displaced.

An exemplary portion of a microarray imaged in accordance with suchconfocal line scanning is illustrated in FIG. 3. Again, referencenumeral 14 refers to the microarray, while reference numeral 32 refersto one of the sample areas in which individual sites 42 are disposed. Inthe illustrated embodiment, the sites are provided in a generallyhexagonal pattern. Scanning by line 34 progresses through successivelines 44 of sites 42. As described in greater detail below, while thepresent confocal line scanning approach may be used with differentlayouts or grid patterns of sites on the micorarray, a hexagonal patternis particularly useful with confocal line scanning insomuch as itprovides for a reduced probability of crosstalk due to the placement andspacing between the sites or site edges. The hexagonal packing,designated generally by reference numeral 46 in FIG. 3, is believed toprovide an optimal degree of accuracy due to such crosstalk reduction,balanced with a superior packing density of the sites.

As described below, and as also illustrated in FIG. 3, as the microarray14 is advanced as indicated by reference numeral 40, the confocalradiation line 34 irradiates a plurality of sites located along theline. The line is wider, in a horizontal direction shown in FIG. 3 thanit is high. Thus, the line may irradiate adjacent sites in a line or rowof sites without irradiating sites in adjacent lines. In a presentembodiment, however, the radiation line 34 is sufficiently thin, at thelevel of the sites, or of a sufficient vertical height in thearrangement illustrated in FIG. 3 to permit it to illuminate less thanthe entire area occupied by the sites. In a presently contemplatedembodiment, the radiation line 34 is, for example, 2 mm in length(horizontal dimension) and less than 3 mm in height (verticaldimension). Thus, the software provided for imaging, mentioned above,may employ techniques such as time delay imaging, in which the readoutfrom the detector described below is shifted with movement of the sampleto provide more accurate representations of the individual sites in eachrow or line.

For purposes of explanation, several aspects of the invention have beenexemplified with regard to moving a microarray past a radiation line. Itwill be understood that embodiments in which the radiation line is movedin addition to or alternatively to moving the microarray can also beused. Thus, line-scanning can be carried out by relative displacement ofa radiation line and/or microarray relative to each other. A portion ofthe sample excited by the radiation line can form a rectangular image onthe detector array (described below).

FIG. 4 is a further diagrammatical representation of the presentconfocal line scanning approach to imaging the microarray 14. Asindicated above, the microarray is radiated along a line 34 as thesupport 30 is slowly moved as indicated by reference numeral 40. Asillustrated in FIG. 4, the line 34 is formed by radiation from a source48 which is directed towards directing optics 50 and therefrom tofocusing optics 52. As described more fully below, the radiation source48 will be a beam with a linear cross section or a radiation lineincluding a plurality of wavelengths of light used to cause fluorescenceat correspondingly different wavelengths from the sample, depending uponthe particular dyes used. The focusing optics 52 will then confocallydirect the radiation line toward the substrate 30 to irradiate the sitesas described above along line 34. It should be noted that the sites maybe provided at the surface of the substrate 30 or slightly below thesurface (e.g., below a protective film or layer). The confocalirradiation along line 34 will essentially focus the radiation towardthe sites themselves at whatever level they are found in the microarray.

The excitation path 54, in the present embodiment, is coplanar with aretrobeam path 56 for radiation returned from the sample by fluorescenceof dyes associated with molecules attached to probes at the individualmicroarray sites. The returned radiation is again focused by focusingoptics 58 such that it impacts a detector 60 to create imaging signalsused to reconstruct an image of the microarray, and of individual siteson the microarray. Specific embodiments for creating the radiation beam,directing the beam to the microarray, and for detecting returnedradiation are described in greater detail below.

It should be noted that, as illustrated generally in FIG. 5, theradiation line used to image the sites simultaneously, in accordancewith the present invention, may be a continuous or discontinuous line.FIG. 5 represents, diagrammatically, a discontinuous line made up of aplurality of confocally directed beams of light which neverthelessirradiate a plurality of points along a line 34. In the embodimentillustrated in FIG. 5, discontinuous beams 62 are created from separatebut adjacent radiation sources 48. These beams, as before, areconfocally directed toward the microarray and irradiate adjacent spots64 along the microarray in a line 34. As with the continuous confocalline scanning described above, the microarray will typically be advancedslowly as indicated by arrow 40 to irradiate successive lines along themicroarray, and thereby successive rows or lines of sites.

Typically, the invention is used to excite and detect a linesimultaneously. In some embodiments, line confocal point scanning can beused such that the optical system directs an excitation point or spotacross a sample by scanning the excitation beam through an objectivelens. The detection system images the emission from the excited point onthe detector without “descanning” the retrobeam. This occurs since theretrobeam is collected by the objective lens and is split off theexcitation beam optical path before returning back through the scanmeans. Therefore the retrobeam will appear on the detector at differentpoints depending on the field angle of the original excitation spot inthe objective lens. The image of the excitation point, at the detector,will appear in the shape of a line as the excitation point is scannedacross the sample. This architecture is useful, for example, if the scanmeans cannot for some reason accept the retrobeam from the sample.Examples are holographic and acoustic optic scan means that are able toscan a beam at very high speeds but utilize diffraction to create thescan. Therefore the scan properties are a function of wavelength. Theretrobeam in fluorescence is at a different wavelength from theexcitation beam.

FIGS. 6 and 7 illustrate an exemplary linearization of an input laserbeam for confocal line scanning of a microarray in accordance with apresently contemplated embodiment. FIG. 6 represents what may beconsidered an elevational view of the conversion or linearization of theinput beam, while FIG. 7 may be considered to illustrate a top planview, although these orientations are understandably interchangeable,depending upon the orientation of the line and microarray to be scanned,as described below. As shown in FIG. 6, an input beam 66 from a laser(not shown) will typically take the form of a circular Gaussian beam 66.An aspherical lens 68, such as a Powell lens converts the input beam toa line 70 of radiation which is directed toward an objective lens 72. Asillustrated in the top view of FIG. 7, the aspherical lens 68effectively produces a generally flat radiation line which is furtherconverted to a confocally concentrated beam 74 by the objective lens 72.

As illustrated in FIG. 8, the arrangement shown in FIGS. 6 and 7produces a linear region of radiation which can be used tosimultaneously irradiate a number of sites on the microarray. FIG. 8 isa graphical representation of the simulated illumination along aradiation line produced by an aspherical lens as described withreference to FIGS. 6 and 7. The relative illumination of the beam isindicated by vertical axis 76, while the image coordinate in millimetersis represented by the horizontal axis 78. In the illustrated embodiment,the illumination intensity rises rapidly near an edge of the asphericallens, as indicated by reference numeral 80 and drops rapidly near anopposite edge, as indicated by reference numeral 82. Between the edges auseful segment of radiation 84 has a substantially constant relativeillumination level. In a present embodiment, the useful width 86 of theradiation line is used to irradiate lines or rows of sites on themicroarray simultaneously. The simulation illustrated in FIG. 8, forexample, provided a useful scanning length 86 of approximately 1.024millimeters, although a number of factors, including the optics involvedmay provide for other useful radiation line lengths.

As will be appreciated by those skilled in the art, for imaging at aplurality of wavelengths, a confocal line scanning fluorescence imagingsystem in accordance with the present technique will provide for linesof multiple wavelengths with the diffraction-limited width and uniformdistribution along a length to irradiate sample sites and thereby toexcite multiple fluorescent dyes. The line generator approachillustrated in FIGS. 6, 7 and 8 provide an exemplary mechanism for suchlinearization of irradiating, multiple wavelength light. The provisionof multiple wavelengths in the radiation line will be described ingreater detail below. Effectively, the arrangement illustrated in FIG.6, 7 and 8 fan a collimated input beam in one dimension and maintain thebeam collimated in a perpendicular dimension. The beam is then focusedby the objective lens 72 to a diffraction-limited line on a focal planeof the lens.

Based upon the sag of the aspherical lens, a collimated pure Gaussianinput beam with a defined beam diameter is preferred to generate a lineof uniform distribution. A presently contemplated technique forobtaining a beam with an almost pure Gaussian distribution is the use ofa single mode fiber or fiber cable to provide input to the asphericallens.

Several arrangements may be foreseen for use of such a single mode fiberor fiber cable. FIG. 9 illustrates a first exemplary embodiment in whicha linear radiation source 88 includes a laser 90 coupled to a singlemode fiber pigtail 92 and therethrough to a line generator module 94.The objective lens downstream of the aspherical lens is omitted from theillustration in FIG. 9. The generated line profile is not only sensitiveto the input beam profile but also sensitive to input beam diameter,collimation characteristics and centering of the beam to the asphericallens. That is, the aspherical lens may be designed for a defined inputbeam diameter, and the assembly, particularly the components of the linegenerator module 94, is aligned to achieve the design performance.

In the illustrated embodiment, the line generator 94 includes severaloptical components which are pre-aligned in a modularized assembly tofacilitate both their quality control and packaging in the scanner. Inparticular, line generator modular 94 may include a collimator 96 thatcollimates, the input beam from the single mode fiber 92 and directs thecollimated beam to an aspherical lens 100. A laser line filter 98 mayalso be employed, particularly for applications of fluorescence imaging,to reduce background noise. The illustration of FIG. 9 may provide forpre-assembling or terminating the single mode fiber 92 on both ends,that is, at the laser 90 and at the line generator module 94.

Alternatively, the linear radiation source 88 may provide for splicing apair of fiber pigtails as illustrated generally in FIG. 10. In theembodiment of FIG. 10, the fiber pigtail 92 is pre-coupled to the laser90, while a second fiber pigtail 102 is pre-coupled to the linegenerator module 94. The two fibers may then be connected or spliced atan intermediate point as indicated generally by reference numeral 104.

In a further alternative configuration, illustrated in FIG. 1, a singlefiber pigtail 102 may again be used, which may be pre-assembled with theline generator module 94. In this embodiment, however, the laser 90provides input to the fiber pigtail 92 by active coupling, as indicatedby reference numeral 106.

In a further alternative configuration, illustrated generally in FIG.12, a fiber pigtail 102 may be pre-assembled with laser 90. Rather thanproviding a collimator in the line generator module 94 as describedabove, a variable beam expander 108 may be employed for providing inputto a modified module 110 which includes an aspherical lens, as before.The embodiment of FIG. 12 may require that the input beam diameter matchthe desired diameter by virtue of the variable beam expander 108.

An exemplary line generator module 94 is illustrated generally in FIG.13. As indicated above, and as shown in the physical implementation ofFIG. 13, the module 94 may receive an input beam, designated generallyby reference numeral 112, via a single mode fiber 92. An outputradiation line 114 is emitted by the module. In the illustratedembodiment, a fiber optic connector 116 serves to join the single modefiber 92 to the input side of the module 94. Therefrom, the beampropagates through collimator 96, laser line filter 98 (where provided),and aspherical lens 100. Again, the modularization of the opticalcomponents used to convert the output of the laser to a radiation lineis favored insomuch as it facilitates assembly of the overall system,alignment of the optics, and later servicing and replacement of theoptical components, if needed.

As indicated above, in certain contemplated embodiments, the radiationsource is a laser. Other useful radiation sources might include, forexample, a lamp such as an arc lamp, quartz halogen lamp and lightemitting diodes. Any of a variety of other radiation sources can be usedas desired for exciting a sample at a particular wavelength. As desiredfor a particular application, the radiation source can generateradiation at various wavelengths including, for example, a wavelength inthe UV, VIS or IR range. For example, an apparatus of the invention caninclude a laser that generates light at 405 nm, 488 nm, 532 nm or 633nm.

Moreover as noted below, the system can include more than one radiationsource. The multiple radiation sources can be lasers each capable ofgenerating radiation at different wavelengths. The use of multipleradiation sources that generate radiation at different wavelengths canbe useful, for example, in applications wherein a sample includes one ormore fluorophores that produce different emission signals when excitedat different wavelengths. Different emission signals can be collectedsimultaneously, for example, using multiple detection arms as set forthbelow in further detail. Alternatively or additionally, differentemission signals can be collected sequentially following sequentialexcitation at different wavelengths.

As noted above, certain embodiments of the invention may further includean expander positioned to receive excitation radiation from a radiationsource and to send an expanded beam of the radiation to a linegenerator. In particular embodiments, the diameter of the excitationbeam generated by the radiation source is approximately 1 mm indiameter. A first expander is capable of expanding the diameter of thebeam. For example, according to one embodiment, the expander expands theexcitation beam to a diameter of 4 mm. Other useful beam expanders canbring the diameter of a radiation beam to at least about 0.5 mm, 1 mm, 2mm, 5 mm, 10 mm, 15 mm, 20 mm or more.

As also discussed above a line generator useful in the invention caninclude a diffractive element configured to generate adiffraction-limited line with uniform intensity distribution. Forexample a cylindrical micro-lens array and a condenser can be used. Thecylindrical micro-lens array can be configured to focus excitationradiation onto the front focal plane of the condenser to generate adiffraction-limited line with uniform intensity distribution. A furtherexample of a line generator is a one-dimensional diffuser having anangular uniformity and a condenser, wherein the one-dimensional diffuseris placed at the front focal plane of the condenser to generate adiffraction-limited line with uniform intensity distribution. Ifdesired, the line generator can further include an aspheric refractivelens to generate a diffraction-limited line with uniform intensitydistribution. An exemplary aspheric refractive lens is a Powell lens.

In a particular embodiment, the line generator can be configured toreceive an input excitation beam having a diameter of 4 mm to obtain afan angle of 6 degrees. Other useful configurations include, but are notlimited to, those that receive an input excitation beam having adiameter of at most about 0.1 to 50 mm. A line generator can obtain afan angle of at least about 0.1° to at most about 80°, full width. Thebeam diameter and fan angle can be selected to achieve a desired shapefor a radiation line. Generally, the width of the radiation line dependsupon beam diameter such that a larger beam diameter provides a widerradiation line in the vertical dimension and the length of the radiationline depends on the fan angle such that a larger fan angle provides alonger radiation line in the horizontal dimension. Typically, the lineshould appear to originate at the pupil of the objective, however thisis not a requirement.

As set forth above, any of a variety of optical elements capable ofgenerating a line can be placed in the optical path between a radiationsource and a sample region to be irradiated. For example, an arc lampfocused on a slit and then collimated can be used to generate a line. Afurther example is an edge emitting diode laser having an anamorphicbeam which generates a line when focused. It will be understood that aradiation source used to irradiate a sample region can itself be capableof generating a line. Thus, a radiation source useful in the inventioncan include a line generator.

Any of a variety of methods and apparatus including, but not limited tothose exemplified above, can be used to direct a radiation line to asample region. The dimensions of the radiation line can be selected toachieve confocality in a single axis of a rectangular detector array.More specifically, the vertical dimension of the radiation line can beshort enough to achieve confocality in the vertical dimension of therectangular detector array.

A line generator of the invention is typically configured to produce aradiation line having a shape at a sample region that is rectangular oroblong. Exemplary shapes include, but are not limited to, a rectangular,elliptical, or oval shape. A line generator can be configured to producea radiation line having one or more of the properties set forth below.

A radiation line that contacts a sample region can have a ratio of the1/e² width of the vertical dimension for the radiation line to thequotient of the vertical dimension for the rectangular detector arraydivided by the magnification of the imaging optics that results inconfocality in one dimension. For example, the ratio can be at leastabout 0.5, 1, 1.5, 2, 3 or higher. An apparatus of the invention can beconfigured to have an upper end for the ratio that is at most about 2,1.5, 1, 0.5 or lower. The ratio can be outside or inside the aboveranges as desired including, for example, being in the range of 0.5 to3.

A radiation line that contacts a sample region can have a ratio of thevertical dimension for the radiation line to the quotient of thevertical dimension for the rectangular detector array divided by themagnification of the imaging optics that results in confocality in onedimension. For example, the ratio can be at least about 0.1, 0.5, 1, 5,10 or higher. The upper end of the ratio can be at most about 10, 5, 1,0.5, 0.1 or lower. The ratio can be outside or inside the above rangesas desired including, for example, being in the range of 0.1 to 10.

Furthermore, the ratio of the vertical dimension for the radiation lineto the Rayleigh resolution of the imaging optics can be at least about0.1, 0.5 1, 5, 10 or higher. The upper end of the ratio can be at mostabout 10, 5, 1, 0.5, 0.1 or lower. The ratio can be outside or insidethe above ranges as desired including, for example, being in the rangeof 0.1 to 10.

Although the invention is exemplified herein with regard to embodimentsin which a sample region is contacted with a radiation line, it will beunderstood that the radiation that contacts a sample region can haveother shapes including, for example, a square or circle.

As described below, an apparatus of the invention can include anobjective positioned to receive radiation therethrough to illuminate asample region. The objective can be further positioned to collectradiation emanating from a sample region and direct it to a detectorarray. Optionally, the apparatus can include a second expanderpositioned to receive the excitation radiation from the line generatorand send an expanded beam of the radiation to the objective. The secondexpander can be further configured to decrease the field angle of theradiation line. For example, after the excitation beam passes throughthe line generator and/or a second expander, it may be directed to anobjective by a beam splitter. In particular embodiments, the objectivehas an external pupil positioned to receive the radiation linetherethrough to illuminate the sample region. Preferably, the beamsplitter may be located near the entrance pupil of the objective lens.The beam splitter can be placed at an axial or lateral position relativeto the objective. If desired, an objective can have a property of colorcorrection, high numerical aperture, telecentricity, afocality at thebackplane or a combination of such properties.

The beam splitter directs the radiation line to an objective. Theobjective can be a microscope objective. The objective may have a focallength of 20 mm. Accordingly, the objective may possess a numericalaperture of 0.366. Further, the objective may have a field angle of ±3degrees and an entrance pupil having a 16 mm diameter. Preferably, theobjective is telecentric. Exemplary telecentric objective lenses usefulin the invention include those that are described in U.S. Pat. No.5,847,400, which is incorporated herein by reference.

FIG. 14 illustrates an overall optical layout for the various componentsdescribed above in a multiple wavelength scanner 1 18. The scanner 1 18may include a plurality of laser light sources, with two such sourcesbeing illustrated in the embodiment of FIG. 14. These include a firstlaser 120 and a second laser 122. The first laser 120, in presentlycontemplated embodiments may be a 658 nm laser, a 750 nm laser, or a 635nm laser, depending upon the desired application. The second laser 122may be, for example, a 488 nm laser, a 594 nm laser, or a 532 nm laser.Other wavelength lasers may, of course, be used. In the presentembodiment, the first laser 120 is a 635 nm laser when the second laser122 is a 488 nm laser, or the first laser 120 is a 750 nm laser when thesecond laser 122 is a 594 nm laser, or the first laser 120 is a 658 nmlaser when the second laser 122 is a 532 nm laser. The selection of thewavelength for each laser will depend, of course, upon the fluorescenceproperties of the dyes used in the microarray, although the wavelengthsof the lasers used in unison for any particular imaging sequence will bedistinct from one another to permit differentiation of the dyes at thevarious sites of the microarray.

Each laser 120 and 122 is coupled to a single mode fiber 124 and 126,respectively, as described above. Moreover, each fiber 124 and 126 feedsa line generator module 94 of the type described above. Downstream ofeach module 94, a filter wheel 128 and 130 may be provided. The filterwheels serve to block, pass or attenuate the light depending upon thedesired function.

Output from each of the lasers 120 and 122 will be converted to a nearpure Gaussian distribution by the respective single mode fibers 124 and126, and the resulting beams will be converted to beams with linearcross-sections, also referred to as radiation lines, by the linegenerator modules 94. Downstream of the filter wheels 128 and 130, thetwo radiation lines will be combined by a beam combiner 132. Thecombined radiation line 134 will, then, comprise light at two differentwavelengths for irradiating the microarray. The combined radiation line134 is then directed to a dichroic beam splitter 136 which directs thebeam toward focusing optics 138. The focusing optics 138 constitute amicroscope objective that confocally directs and concentrates theradiation line along the line to the microarray 14 as described above.Although the invention is exemplified herein with regard to a combinedradiation line that forms a single radiation line it will be understoodthat the two radiation lines can be combined such that two lines arenearly collinear. Thus, a portion of the microarray that is irradiatedwith the combined radiation line will be irradiated with the nearlycollinear lines of radiation. The two lines are typically separated by adistance equivalent to the width of each line in order to minimizecrosstalk between channels.

As illustrated diagrammatically in FIG. 14, the microarray 14 may besupported on a stage that allows for proper focusing and movement of themicroarray before and during imaging. The stage can be configured tomove the sample, thereby changing the relative positions of therectangular image and the rectangular detector array in the scan-axis(vertical) dimension. Movement of the translation stage can be in one ormore dimensions including, for example, one or both of the dimensionsthat are orthogonal to the direction of propagation for the radiationline and typically denoted as the x and y dimensions. In particularembodiments, the translation stage can be configured to move in thedirection perpendicular to the scan axis for a detector array. A stageuseful in the invention can be further configured for movement in thedimension along which the radiation line propagates, typically denotedas the Z dimension. Movement in the Z dimension can be useful forfocusing the apparatus. In the configuration of FIG. 14, the stagecomponent include tilt actuators 140, typically used for focusing theradiation line, Y-direction actuators and eject components 142 forplacing the microarray in a position for scanning, and for grossmovements of the microarray between scans, and an X-direction actuators144 for fine movements of the microarray during scanning.

Sites on the microarray 14 may fluoresce at wavelengths corresponding tothose of the excitation beam and return radiation for imaging. As willbe appreciated by those skilled in the art, the wavelength at which thedyes of the sample are excited and the wavelength at which theyfluoresce will depend upon the absorption and emission spectra of thespecific dyes. Such returned radiation will propagate through beamsplitter 136 as indicated generally by retrobeam 146 in FIG. 14. Thisretrobeam will generally be directed toward one or more detectors forimaging purposes. In the illustrated embodiment, for example, the beamis directed toward a mirror 148 and therefrom to a second dichroic beamsplitter 150. A portion of the beam, as indicated by reference numeral154, is then directed by mirrors 152 to a bandpass filter wheel 158 thatfilters the beam to obtain the desired output wavelength correspondingto one of the fluorescent dyes of the sites in the microarray. Inparticular embodiments, the portions of the beam that are directed todifferent mirrors can be the respective lines of a combined beam thatforms two nearly co-linear lines. A projection lens 160 then directs thefiltered beam to a charge coupled device (CCD) sensor 164 which producesoutput signals corresponding to locations of the radiation in thereceived beam. Similarly, a second portion 156 of the beam from beamsplitter 150 is directed to another mirror through a different bandpassfilter wheel 158 and projection lens 160. The second beam 156 may alsobe directed through an optional chromatic aberration compensation device162, which may be motorized. The chromatic aberration compensationdevice 162 serves to bring both wavelength channels into co-focus.Finally, beam 156, filtered and focused by filter wheel 158 and lens 160is directed to a second CCD sensor 166. The receipt and processing ofsignals from the sensors 154 and 166 may be managed by a control board168.

A rectangular detector array of the invention can be configured to forma virtual slit as set forth previously herein. In particularembodiments, the size and dimensions of the virtual slit can bedetermined from the ratio of the vertical dimension for the rectangulardetector array and the product of the Rayleigh resolution of the imagingoptics multiplied by the magnification of the imaging optics. Forexample, the ratio of the vertical dimension for the rectangulardetector array and the product of the Rayleigh resolution of the imagingoptics multiplied by the magnification of the imaging optics can be inthe range of 0.1 to 10 or in the range of 0.5 to 3. An apparatus of theinvention can be configured to obtain an image of a sample at a desiredor optimal Rayleigh resolution including, for example, a Rayleighresolution between 0.2 and 10 micrometers.

In particular embodiments, the aspect ratio of the number of detectionelements in a first dimension to the number of detection elements in thescan-axis dimension for a rectangular detector array can be greater than2, 10, 20, 50, 100, 1000 or higher. For example, a line scan CCD cameracan be configured to capture, four thousand (4,000) pixels in the firstdimension and n pixels in the scan-axis (vertical) dimension. The CCDline scan camera can be designed such that the resolution along thelength of the line is matched to the system resolution. In this case,the horizontal axis includes approximately 4,000 CCD elements along thelength of a 2 mm radiation line, resulting in a 0.5 μm pixel resolutionat the object. The number of CCD elements “n” in the directionperpendicular to the horizontal axis, also referred to as the verticalaxis, can be chosen to collect substantially all of the emittedradiation while reducing the amount of background radiation collected.According to one embodiment of the invention, the CCD has 4096 pixels,each 12 μm in size. To image a 2 mm line to this size CCD requires amagnification of 25×. Accordingly, n can be in the range of six to eightpixels. The design architecture limits the excitation error in theconfocal axis such that predominantly 100% of the excitation radiationis contained within a spot comparable with the resolution of the system.In this case, the spot size would be roughly 1.0 μm.

Although the apparatus has been exemplified above with regard to a CCDline scan camera, it will be understood that any of a variety of otherdetectors can be used including, but not limited to a detector arrayconfigured for TDI operation, a CMOS detector, APD detector, Geiger-modephoton counter or other detector set forth elsewhere herein.

In general, the operation of the various components illustrated in FIG.14 may be coordinated by system controller 170. In a practicalapplication, the system controller will include hardware, firmware andsoftware designed to control operation of the lasers, movement andfocusing of the objective 138 and microarray support, and theacquisition and processing of signals from the sensors 164 and 166. Thesystem controller may thus store processed data, and further process thedata for generating a reconstructed image of the irradiated sites thatfluoresce on the microarray.

FIG. 15 illustrates an alternative arrangement for the multiplewavelength scanner, designated generally by reference numeral 172. Inthis alternative arrangement, beams from separate lasers are combinedand the cross section of the combined beam then converted to a linearshape by an aspherical lens. Thus, as in the previous embodimentsummarized with reference to FIG. 14, input lasers 120 and 122 providewavelengths of light corresponding to dyes used at various sites on amicroarray 14. In the embodiment 172, however, a first laser 120 outputsits beam to a single mode fiber 124, followed by a collimator 174 thatcollimates this output. The collimated output may then be directed to afilter wheel 130, and the resulting beam 176 is directed, by mirrors 152to a variable beam expander 180 of the type described above withreference to FIG. 12.

Similarly, output from the second laser 122 is directed through a secondfilter wheel 130 and the resulting beam 178 is directed, such as viamirrors 152 to a second variable beam expander 182. Output from thevariable beam expanders, then, is joined by a beam combiner 132. Thecombined beam 182, which will include light at the desired wavelengthsfor radiation of the microarray is converted to a line by an asphericallens 100. As before, then, a combined radiation line 134 including lightat the desired wavelengths will be produced and directed to themicroarray 14 by a beam splitter 136. The remaining components of thesystem may be essentially identical to those described above withrespect to FIG. 14.

FIG. 16 provides a somewhat more detailed opto-mechanical diagrammaticalrepresentation of a multiple wave-length scanner in accordance withaspects of a presently contemplated embodiment. The scanner 184 mayinclude a first laser assembly 186 which, itself, includes multiplelasers. In the illustrated embodiment, for example, laser assembly 186includes a first laser 188 which may be a 488 nm laser, and a secondlaser 190 which may be a 658 nm laser. The system may further include asecond laser assembly 192, which may include, for example, a 594 nmlaser 194 and a 750 nm laser 196. As will be appreciated by thoseskilled in the art, the inclusion of multiple laser assemblies 190 and192 may allow for different types of scanning operations to be performedwith a single scanner, such as decoding functions, analytical functions,and so forth. For example, lasers 188 and 190 may be used in conjunctionwith one another for certain types of decoding operations, while lasers194 and 196 may be used in conjunction with one another for other typesof decoding. The assemblies may include other lasers which mayalternatively be used, or other assemblies may be provided, such as anassembly employing a 635 nm laser and a 532 nm laser, such as forcertain analytical operations.

The laser assemblies 190 and 192 are coupled to single mode fibers 122and 124 that, as described above, convert the output of the lasers tonear pure Gaussian distributions. The light transmitted via the fibers122 and 124 is input to line generator modules 94 to produce radiationlines. The beams of radiation are then directed to excitation filters128, and combined by combiner 132 to form a combined radiation line 134.A filter wheel 130 may filter this combined radiation line, such as toblock, pass or attenuate the beam as desired.

As in the embodiments described above, the filtered combined radiationline is then directed to a beam splitter 136 and therefrom to anobjective 138. In the embodiment illustrated in FIG. 16, the objectiveis provided with an autofocus system 198 that may include one or moreactuators, such as a voice coil, a linear motor stage, a piezo motorstage, or a piezo flexure stage. Sensors 200 provide for sensing thedistance or focus of the system on the microarray 14, and serve toprovide feedback for dynamic focusing of the confocally-directedradiation line on the appropriate depth along the microarray 14.

FIG. 16 also provides somewhat more detail regarding a presentlycontemplated arrangement for moving the microarray 14 prior and duringscanning. For example, a sample handling tray 202 is provided along witha motor 204 for moving the tray in and out of an imaging position. Anadapter plate 206 allows for positioning of the microarray in a dockingstation 208. Actuators 210 provide for appropriate positioning of themicroarray in the docking station. A coarse stage 212, controlled by astepper motor 214 allows for coarse control of the position of themicroarray with respect to the combined radiation line confocallydirected toward the microarray. The coarse stage 212 may, for example,be used to appropriately position a portion of the microarray on whichthe sites are located that are to be imaged. A precision stage 216,which may include a linear motor 218 and a linear encoder 220 serve toprovide for fine positioning and movement of the microarray prior to andduring scanning.

As before, radiation resulting from fluorescence of individual sites onthe microarray is returned through the beam splitter 136 to mirrors orother optical devices used to direct the retrobeam through bandpassfilters 158, projection lenses 160 and ultimately to CCD sensors 164 and166.

The foregoing arrangements provide for extremely rapid and accurateimaging of multiple sites on a microarray by use of a radiation linethat excites the sites simultaneously. It has been found that theconfocal line scanning technique of the present invention isparticularly useful in applications where sites on the microarray arespaced from one another such as to, in combination with the linearscanning described above, reduce the potential for crosstalk betweenreturned radiation from the individual sites. FIG. 17 illustrates apresently contemplated arrangement of sites in a hexagonal grid array totake advantage of this aspect of the confocal line scanning technique ofthe invention.

As illustrated in FIG. 17, an array section 222 will include a pluralityof sites 42 provided in a predetermined pattern. A presentlycontemplated embodiment provides a hexagonal packing pattern asillustrated. The pattern includes what may be termed adjacent rows orlines of sites designated by reference numerals 224 and 226 in FIG. 17.As will be appreciated by those skilled in the art, the orientation ofthe lines may generally be thought of with reference to the direction ofscanning by the confocally directed radiation line described above. Asradiation is directed along lines parallel to the site lines 224 and226, then, a portion of the lines of sites will be illuminated by theradiation, and return a retrobeam which will be bright in those areasthat fluoresce. Adjacent sites 228 and 230 in each row or line of siteswill be spaced from one another, and both of these sites will be spacedfrom a nearest adjacent site, such as site 232 of an adjacent row orline 226. The distance between successive or adjacent lines of sites maybe designated generally by reference numeral 234, such as by referenceto the center of the sites in each line. It will be noted that with thehexagonal packing pattern of FIG. 17, the distance between the centersof adjacent sites in the same line, however, is greater than thedistance between the adjacent lines of sites. Moreover, in theorientation of FIG. 17, the distance between centers of adjacent sitesin the same line is greater than the nearest distance 236 between sitesin the adjacent lines. In particular, for a hexagonal packing pattern ofthe type illustrated in FIG. 17, distance 234 will be approximately0.866 (the cosine of 60 degrees) of the distance 236.

Moreover, if the sites 228, 230 and 232 are considered to have edges238, these edges will be spaced from one another by a distance greaterthan would result if the sites were disposed in a rectilinear pattern.That is, the projection of the distance between the edges 238 of sites228 and 232 along the axis of scanning may be denoted by referencenumeral 240. The actual distance, however, between the edges will begreater, as indicated by reference numeral 242. Again, for the hexagonalpattern illustrated in FIG. 17, the distance 242 will be approximately15% greater than the distance 240.

As will be appreciated by those skilled in the art, as the density ofthe sites on microarrays is increased, and spacing between the sites isconsequently decreased, increasing demands are made on the ability tocarefully focus the irradiating light beam on the sites, and to properlyfocus the retrobeam for imaging purposes. The present technique providesexcellent results in the ability to confocally irradiate a line ofsites, where the confocality exists in the axis parallel to the width ofthe radiation line and not along the length of the radiation line.However, crosstalk between the sites may be considered as a relativeinability to distinguish between the sites, as the images produced fromhigh intensity sites spills over in the nonconfocal axis to neighboringsites. This can be, problematic, for example, when high intensity sitesare located immediately adjacent to very low intensity sites. Thecombination of confocal line scanning with non-rectilinearly packedsites, in particularly in combination with hexagonally packed sites isbelieved to provide far superior distinction between irradiated andimaged sites, due to the reduction in crosstalk and blurring between theimaged sites.

The combination of a hexagonal arrangement of sites and the radiationline orientation set forth above is one example of an embodiment of theinvention wherein the distances between nearest neighbor sites that areirradiated simultaneously by a radiation line at a first scan positionis greater than the distance between nearest neighbor sites that areirradiated at different times by the scanning radiation line. It will beunderstood that other combinations of site packing and line orientationcan also be used to achieve similar advantages. For example, althoughcircular sites in a rectilinear grid are not packed as closely as in ahexagonal grid, the orientation for a radiation line and its directionof scan can be selected for a desired reduction in cross-talk. Morespecifically, the radiation line can be oriented diagonally with respectto the rows and columns of sites in the rectilinear grid and theradiation line can be scanned across the grid in the diagonal directionto achieve less cross talk between the sites than if the radiation linewas oriented orthogonally with respect to the rows and columns of sitesin the rectilinear grid and scanned in the orthogonal direction. Anadvantage being that the line is oriented such that the greatest spacingbetween adjacent sites occurs in the nonconfocal axis, parallel to theradiation line.

The packing arrangements described above are particularly useful whenused with a radiation line that is substantially narrower than the widthof the sites being irradiated. In particular embodiments, the width ofthe radiation line (i.e. the shorter dimension of the line) will be atmost 75%, 66%, 50%, 30%, 25% or 10% of the width of the sites beingirradiated. Generally, sites having a regular shape are preferred, forexample, sites having reflectional symmetry or rotational symmetry.However, irregular shaped sites can be used if desired for a particularapplication. Whether a site is regular or irregular in shape the widthfor the site will typically be measured at the widest dimension, forexample, width is measured as the diameter of a site having a circularcross-section.

As illustrated in FIGS. 18-23, a diffraction-limited line with uniformintensity distribution can be generated using a number of architectures.In one such embodiment, shown in FIG. 18, the line generator 244 can beformed with a cylindrical micro-lens array 246 and a condenser 248. Acylindrical micro-lens array 246 is used to focus the excitation beam250 to the front focal plane of a condenser 248 in one dimension whileleaving a second dimension unaffected. A diffraction-limited line 252with uniform intensity distribution will be generated on the back focalplane of the condenser 248. The uniformity of the line is related to thenumber of cylindrical micro-lenses 246 that cover the entrance pupil ofthe condenser 248. The greater the number of cylindrical micro-lensarrays 246, the more uniform the line intensity distribution will be.

According to another embodiment and as shown in FIG. 19, the linegenerator 244 can be formed with a one-dimensional diffuser 254 and acondenser 248. A one-dimensional diffuser 254 having an angularuniformity is placed at the front focal plane of a condenser 248. Thediffuser 254 fans the input collimated beam 250 in one dimension andleaves another dimension unaffected. A diffraction-limited line 252 withuniform intensity distribution will be generated on the back focal planeof the condenser 248. Since the diffuser 254 has angular uniformity, thegenerated line will be uniform.

In still another embodiment of the invention, an objective 256 is usedas a condenser 248. Preferably, the objective lens 256 is a telecentriclens with an external pupil size of 15.75 mm. Preferably, this size isconfigured to match the diameter of the collimated input excitation beam250. In addition, the input field angle of the lens is ±3 degrees, whichcorresponds to a field view of 2 mm.

FIG. 20 shows a one-dimensional diffuser 254 in use with the objective256 described above. As shown in FIG. 20, a one-dimensional diffuser 254is placed at the pupil stop of the objective 256. The objective 256diffuses the collimated input beam 250 to different angles in a certainrange in one dimension and leaves another dimension unaffected. Thediffuser 254 has angular uniformity, i.e. the intensities of beamsdiffused to different angles are the same. The lens 256 focuses the beamat each particular angle to a point in the line. The uniformity of theline is determined by the angular sensitivity of the diffuser 254. Inaddition, the length of radiation line 268 is determined by the fanangle of the diffuser 254. The larger the fan angle is, the longer thegenerated radiation line 268 will be. If the fan angle of the diffuser254 is ±3°, the generated line length will be 2 mm. Although the lengthof the radiation line 268 can be longer than 2 mm, a desired uniformitycan be obtained by a line 2 mm in length.

According to another embodiment, FIG. 21 shows a cylindrical micro-lensarray 246 in use with the above-described objective 256. Eachcylindrical micro-lens 246 samples a portion of the collimated inputbeam 250, focuses it to the pupil stop of the objective 256 in onedimension, and leaves the second dimension unaffected. The cylindricalmicro-lens array 246 fans the beam 250 to different angles in a certainrange in one dimension. The fan angle is determined by the f-number ofthe cylindrical micro-lenses 246. The objective lens 256 focuses thebeam 250 at each angle to a point in the line. Since each point in thefocused line gets contribution from all the cylindrical micro-lenses246, the uniformity of the line is related to the number of cylindricalmicro-lenses 246 that covers the entrance pupil of the objective lens256. For example, according to one embodiment of the invention, 158micro-lenses are used to cover the pupil stop in order to generate auniform line excitation 268.

FIGS. 22 and 23 show additional embodiments of relay telescopes,configured for fluorescent imaging. A relay telescope 258 is positionedbetween the one-dimensional diffuser 254 (see FIG. 22) or cylindricalmicro-lens array 246 (see FIG. 23) and a dichroic beam splitter 260. Thedichroic beam splitter 260 is configured to separate the fluorescenceimaging path (retro-beam) 262 from the excitation path 250.

A CCD camera or other detector array used in the invention can beconfigured for binning. Binning increases the detector array'ssensitivity by summing the charges from multiple pixels in the arrayinto one pixel. Exemplary types of binning that can be used includehorizontal binning, vertical binning, or full binning. With horizontalbinning, pairs of adjacent pixels in each line of a detector array aresummed. With vertical binning, pairs of adjacent pixels from two linesin the array are summed. Full binning is a combination of horizontal andvertical binning in which four adjacent pixels are summed.

Binning in the invention can be carried out with larger sets of sensorelements. As illustrated in FIG. 24( a), the line scan CCD camera andcorresponding control electronics can be configured such that all pixelelements in the vertical axis are collected in a common bin and read outas a single value. Thus, binning need not be limited to adjacent pairsor adjacent groups of array elements. Accordingly, a set of more than 2sensor elements, such as pixels of a CCD camera, can be binned even ifthe set includes non-adjacent sensor elements. Non-adjacent sensorelements occur, for example, in a linear arrangement of 3 sensorelements where the first and third elements are separated from eachother by the intervening second sensor element.

As shown in FIG. 24( b), in binning, all of the pixels in a row areshifted out at once after a single integration time. The advantage ofthis approach, when used in an apparatus of the invention, is thatcompared to a common TDI design the readout rate is less sensitive tojitter. Furthermore, the apparatus would have confocality in one axis,and the tolerance of the synchronization timing of the readout with theY-stage movement would be reduced. FIG. 24( b) shows the projection of a1 μm laser spot on a line scan CCD camera. The projection is symmetricalin both the X and Y-axis. Limiting the number of CCD pixels to 6 in thevertical axis creates a virtual slit in that axis. The same effect canbe achieved with a TDI camera, the main requirement is that the numberof pixels in the vertical axis be optimized to pass a signal while alsorejecting background noise. To achieve this, the laser spot size is setto match the resolution of the system in conjunction with limiting thenumber of vertical pixels.

An alternate embodiment of the invention uses a TDI design which limitsthe number of vertical pixels such that the virtual slit is stillcreated. As shown in FIG. 24( c), in TDI, pixels are shifted in syncwith the encoder output of the y-stage. Additionally, the advantage oversystem designs where n=1 are that the collection efficiency of thesystem would be increased and the sensitivity to small optical alignmentdrifts would be decreased. Exemplary TDI designs and methods that can beused in the invention are described in U.S. Pat. No. 5,754,291, which isincorporated herein by reference.

According to another embodiment of the invention, the present scanningsystem architecture is configured to use parallel multi-spectralfluorescence imaging using line-scan imaging sensors. As shown in FIG.25, radiation line 134 is used to excite fluorescent molecules in a fullspectral range and a chromatic dispersion element 264 is used to spreadthe line fluorescence image 262 across multiple line-scan imagingsensors 266. The system can be implemented using side illumination orcollinear illumination. According to this embodiment of the invention, amulti-band filter set 268 is used to excite and detect multiplefluorescent molecules. As represented in FIG. 26, each of the pluralityof sensors 266 is mapped to a narrow band spectral range. The sensors266 can be imaging sensors such as a linear line-scan CCD or a TDIline-scan CCD. Sensors are also referred to as detectors herein.

As shown in FIG. 27, according to still another embodiment of theinvention, the scanning system architecture can be configured to use amulti-line illumination technique. The system can be implemented usingside illumination or collinear illumination. Here, each line 268 excitesa sample region at a different wavelength, for example, to excitedifferent fluorescent molecules. The resulting multi-line fluorescenceimage is collected by a detector 266 with multiple line-scan imagingsensors 266. Each sensor 266 generates the corresponded fluorescentimage. Because the fluorescence with different spectral ranges isalready spatially separated, no chromatic dispersion element 264 isrequired. A multi-notch filter 270 is used to effectively block residualRayleigh and Raman scattered radiation.

Further, if a chromatic dispersion element is used in the system of FIG.27, images with higher spectral resolution can be collected. Asillustrated in FIG. 28, each sensor group 266 in the figure can alsowork in TDI mode to generate a single integrated image, which providesimages with hierarchical spectral resolution.

The scanning system architecture can be designed to excite fluorescenceof multiple dyes in different spectral ranges simultaneously. Exemplaryarchitectures include a single line with multi-colors used in the systemof FIG. 25 or spaced multi-lines with multi-colors used in the system ofFIG. 27. The radiation source can be a white light lamp with amulti-band excitation filter or a combination of multiple lasers. Theexcitation filter of the multi-band filter set 268 in the system of FIG.25 is not required, for example, if the combination of multiple lasersis used as the radiation source. In addition, the illumination can becollinear illumination (illumination shares the same objective lens 138as the collection) as shown in FIG. 24 or slide illumination (darkfield) as shown in FIG. 28. A multi-band dichroic beam splitter 136(shown in FIG. 25) can be used for the collinear illumination andomitted for the side illumination embodiment. Also as shown in FIG. 25,a multi-band emission filter 272 of the multi band filter set 82 can beused to selectively block excitation radiation while passingfluorescence bands. For illumination with multiple lasers, a multi-notchfilter 270 can also be used to selectively block excitation radiationwhile passing fluorescence bands, which provides even more efficientflorescence detection.

According to particular embodiments of the invention, emission filters272 can be integrated with the image sensor 266. An exemplaryorientation is shown in FIG. 29. A different orientation for blockingmulti-band illumination and multiple laser illumination is shown inFIGS. 30( a) and 30(b) respectively.

An apparatus or method of the invention is particularly useful forobtaining an image of a 2-dimensional area of a sample. Thus, ifdesired, detection can be substantially restricted to obtaining an imagein 2 of the 3 possible dimensions for a sample. Accordingly, an image ofa surface for a sample of interest can be detected or imaged. Aparticularly relevant sample is a microarray. Using the invention thesurface of a microarray can be detected or imaged to determine one ormore property of the microarray. Exemplary properties of a microarraythat can be detected include, but are not limited to, the presence orabsence of a label, the location of a label at a particular locationsuch as a location where a particular probe resides, or a specificcharacteristic of a label such as emission of radiation at a particularwavelength or wavelength range.

Detection of such properties for a microarray can be used to determinethe presence or absence of a particular target molecule in a samplecontacted with the microarray. This can be determined, for example,based on binding of a labeled target analyte to a particular probe ofthe microarray or due to a target-dependent modification of a particularprobe to incorporate, remove or alter a label at the probe location. Anyone of several assays can be used to identify or characterize targetsusing a microarray as described, for example, in U.S. Pat. App. Pub.Nos. 2003/0108867, 2003/0108900, 2003/0170684, 2003/0207295, or2005/0181394, each of which is hereby incorporated by reference.

Exemplary labels that can be detected in accordance with the invention,for example, when present on a microarray include, but are not limitedto, a chromophore; luminophore; fluorophore; optically encodednanoparticles; particles encoded with a diffraction-grating;electrochemiluminescent label such as Ru(bpy)268+; or moiety that can bedetected based on an optical characteristic. Fluorophores that areuseful in the invention include, for example, fluorescent lanthanidecomplexes, including those of Europium and Terbium, fluorescein,rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,methyl-coumarins, pyrene, Malacite green, Cy3, Cy5, stilbene, LuciferYellow, Cascade Blue™, Texas Red, alexa dyes, phycoerythin, bodipy, andothers known in the art such as those described in Haugland, MolecularProbes Handbook, (Eugene, Oreg.) 6th Edition; The Synthegen catalog(Houston, Tex.), Lakowicz, Principles of Fluorescence Spectroscopy, 2ndEd., Plenum Press New York (1999), or WO 98/59066, each of which ishereby incorporated by reference.

Any of a variety of microarrays known in the art, including, forexample, those set forth elsewhere herein, can used as a sample in theinvention. A typical microarray contains sites, sometimes referred to asfeatures, each having a population of probes. The population of probesat each site typically is homogenous, having a single species of probebut in some embodiments the populations can each be heterogeneous. Sitesor features of an array are typically discrete, being separated withspaces between each other. The size of the probe sites and/or spacingbetween the sites can vary such that arrays can be high density, mediumdensity or lower density. High density arrays are characterized ashaving sites separated by less than about 15 μm. Medium density arrayshave sites separated by about 15 to 30 μm, while low density arrays havesites separated by greater than 30 μm. An array useful in the inventioncan have sites that are separated by less than 100 μm, 50 μm, 10 μm, 5μm, 1 μm or 0.5 μm. An apparatus or method of the invention can be usedto image an array at a resolution sufficient to distinguish sites at theabove densities or density ranges.

Although the invention has been exemplified above with regard to the useof a microarray as a sample, it will be understood that other sampleshaving features or sites at the above densities can be imaged at theresolutions set forth above. Other exemplary samples include, but arenot limited to, biological specimens such as cells or tissues,electronic chips such as those used in computer processors, or the like.A microarray or other sample can be placed in a sample region of anapparatus of the invention by being placed on a sample stage such asthose described elsewhere herein.

An apparatus of the invention can further include a processor, operablycoupled to a rectangular detector array or otherwise configured toobtain data from the rectangular detector array, wherein the processoris configured to perform a plurality of functions on the image. Theprocessor can include a conventional or general purpose computer systemthat is programmed with, or otherwise has access to, one or more programmodules involved in the analysis of imaging data. Exemplary computersystems that are useful in the invention include, but are not limited topersonal computer systems, such as those based on Intel®, IBM®, orMotorola® microprocessors; or work stations such as a SPARC® workstationor UNIX® workstation. Useful systems include those using the Microsoft®Windows®, UNIX or LINUX® operating system. The systems and methodsdescribed herein can also be implemented to run on client-server systemsor wide-area networks such as the Internet.

The processor can be included in a computer system, configured tooperate as either a client or server. The processor can executeinstructions included in one or more program modules. Results from oneor more program modules such as an image of a sample or sample region,or analysis of the sample or sample region can be reported to a user viaa graphical user interface. For example, results can be reported via amonitor or printing device operably connected to the processor. Thus, animage of an array or other sample can be provided to a user via agraphical user interface.

According to certain aspects of the invention, several advantages arerealized. The system of the present invention scans samples faster thanother technologies and provides improved data quality at lower cost.Specifically, the readout rate of the present invention is increased bya factor of n as compared to conventional TDI systems. Confocality canbe achieved in one or more axis. In addition, the present invention isless sensitive to optical alignment drifts.

Further, the present invention combines the advantages of simultaneousexcitation/detection of multiple fluorescent molecules using multi-bandfilters and parallel readout of multiple line-scan imaging sensors onthe same sample. The present invention can simultaneously generatemulti-spectral fluorescence images in a fast speed. In particularembodiments, an apparatus of method of the invention can scan a sampleat a rate of at least about 0.01 mm²/sec. Depending upon the particularapplication of the invention faster scan rates can also be usedincluding, for example, in terms of the area scanned, a rate of at leastabout 0.02 mm²/sec, 0.05 mm²/sec, 0.1 mm²/sec, 1 mm²/sec, 1.5 mm²/sec, 5mm²/sec, 10 mm²/sec, 50 mm²/sec or 100 mm²/sec or faster. If desired,for example, to reduce noise, scan rate can have an upper limit of about0.05 mm²/sec, 0.1 mm²/sec, 1 mm²/sec, 1.5 mm²/sec, 5 mm²/sec, 10mm²/sec, 50 mm²/sec or 100 mm²/sec. Scan rate can also be measured interms of the rate of relative movement for an image and detector in thescan-axis (vertical) dimension and can be, for example, at least about0.1 mm/sec, 0.5 mm/sec, 1 mm/sec, 10 mm/sec, or 100 mm/sec. Again, toreduce noise, scan rate can have an upper limit of about 0.5 mm/sec, 1mm/sec, 10 mm/sec, or 100 mm/sec. In sum, the present invention can beused to build multi-spectral fluorescence imagers, which are moreefficient and cost-effective than other imaging systems.

The following are terms that are used in the present discussion, andwhich are intended to have the meanings ascribed below.

As used herein, the term “radiation source” is intended to mean anorigin or generator of propagated electromagnetic energy. The term caninclude an illumination source producing electromagnetic radiation inthe ultra violet (UV) range (about 200 to 390 nm), visible (VIS) range(about 390 to 770 nm), or infrared (IR) range (about 0.77 to 25microns), or other range of the electromagnetic spectrum. A radiationsource can include, for example, a lamp such as an arc lamp or quartzhalogen lamp, or a laser such as a solid state laser or a gas laser oran LED such as an LED/single mode fiber system.

As used herein, the term “excitation radiation” is intended to meanelectromagnetic energy propagated toward a sample or sample region.Excitation radiation can be in a form to induce any of a variety ofresponses from a sample including, but not limited to, absorption ofenergy, reflection, fluorescence emission or luminescence.

As used herein, the term “sample region” is intended to mean a locationthat is to be detected. The location can be, for example, in, on orproximal to a support device that is configured to support or contain anobject to be detected. A sample can occupy a sample region permanentlyor temporarily such that the sample can be removed from the sampleregion.: For example a sample region can be a location on or near atranslation stage, the location being occupied by a microarray whenplaced on the translation stage.

As used herein, the term “detector array” is intended to mean a deviceor apparatus having several elements that convert the energy ofcontacted photons into an electrical response. An exemplary detectorarray is a charge coupled device (CCD), wherein the elements arephotosensitive charge collection sites that accumulate charge inresponse to impinging photons. Further examples of detector arraysinclude, without limitation, a complementary metal oxide semiconductor(CMOS) detector array, avalanche photodiode (APD) detector array, or aGeiger-mode photon counter detector array. The elements of a detectorarray can have any of a variety of arrangements. For example, arectangular detector array has elements in a 2-dimensional, orthogonalarrangement in which a first dimension, referred to as the “horizontal”dimension is longer than a second dimension referred to as the“vertical” dimension. A square detector array has elements in a2-dimensional, orthogonal arrangement in which the first and seconddimensions of the arrangement are the same length.

As used herein, the term “rectangular image” is intended to mean anoptically formed representation of a sample, or portion of the sample,that occurs within a 2-dimensional, orthogonal region having ahorizontal dimension that is longer than the vertical dimension. Therectangular image can represent the entirety of an image emanating froma sample region or, alternatively, can be a rectangular portion of alarger image, the larger image having any of a variety of shapes.

As used herein, the term “scanning device” is intended to mean a devicecapable of sequentially detecting different portions of a sample. Ascanning device can operate, by changing the position of one or morecomponent of a detection apparatus including, for example, a sample,radiation source, optical device that directs excitation radiation to asample, optical device that directs radiation emanating from a sample,or detector array. Exemplary scanning devices include, but are notlimited to a galvanometer configured to move a beam or line of radiationacross a sample or a translation stage configured to move a sampleacross a beam or line of radiation.

As used herein, the term “Rayleigh resolution” is RR in the followingequation

RR=((1.22)(λ)(f))/D

wherein λ is wavelength, f is focal length and D is distance between twoobjects that are detected. The term is intended to be consistent withits use in the art of optics, for example, as set forth in Hecht,Optics, 4th ed., Addison Wesley, Boston Mass. (2001), which is herebyincorporated by reference.

As used herein, the term “magnification” is intended to mean the ratioof the size of an object to the size of an image of the object. Forexample, magnification can be determined from the ratio of the size ofsample region (i.e. the object) to the size of an image of the sampleregion at a detector array. In systems including an objective andprojection lens, magnification can be determined from the ratio of focallength of the objective to back focal length of the projection lens.

As used herein, the term “radiation line” is intended to mean acollection of electromagnetic waves or particles propagated in a uniformdirection, wherein the 2-dimensional cross section orthogonal to thedirection of propagation is rectangular or oblong. Exemplary2-dimensional cross sections of a radiation line include, but are notlimited to, a rectangular, elliptical, or oval shape. The crosssectional width of a radiation line can have one or both dimensions in arange of, for example, about 0.05 μm to about 10 μm. For example, adimension of the radiation line can be at least about 0.05 μm, 0.1 μm,0.5 μm, 1 μm, 5 μm or 10 μm. Furthermore, a dimension of a radiationline can be, for example, at most about 0.1 μm, 0.5 μm, 1 μm, 5 μm or 10μm. It will be understood that these dimensions are merely exemplary andradiation lines having other dimensions can be used if desired.

As used herein, the term “line generator” is intended to mean an opticalelement that is configured to generate a diffraction-limited or neardiffraction-limited radiation line in the plane perpendicular to theoptical axis of propagation with a substantially uniform intensitydistribution along the horizontal axis of the line. Exemplary linegenerators include, but are not limited to, a one dimensional diffuserhaving angular uniformity, cylindrical microlens array, diffractiveelement or aspheric refractive lens such as a Powell lens. The onedimensional diffuser having angular uniformity or cylindrical microlensarray can be placed to direct radiation to a condenser.

As used herein, the term “beam splitter” is intended to mean an opticalelement that passes a first portion of a radiation beam and reflects asecond portion of the beam. For example a beam splitter can beconfigured to selectively pass radiation in a first wavelength range andreflect radiation in a second, different radiation range. When used forfluorescence detection the beam splitter will typically reflect theshorter wavelength excitation radiation and transmit the longerwavelength emission radiation.

As used herein, the term “external pupil” is used in reference to anobjective, where the entrance pupil to the back aperture of theobjective is behind the physical dimensions of the objective in theexcitation beam path.

As used herein, the term “expander” is intended to mean one or moreoptical elements configured to adjust the diameter and collimation of aradiation beam. For example, an expander can be configured to increasethe diameter of a radiation beam by a desired amount such as at least 2fold, 5 fold, 10 fold or more. Optical elements of an expander caninclude, for example, one or more mirrors or lenses.

As used herein, the term “projection lens” is intended to mean anoptical element configured to transfer the image of an object to adetector. For example, a lens can be placed to transfer an imageemanating from an objective lens to a detector array.

As used herein, the term “optical filter” is intended to mean a devicefor selectively passing or rejecting passage of radiation in awavelength, polarization or frequency dependent manner. The term caninclude an interference filter in which multiple layers of dielectricmaterials pass or reflect radiation according to constructive ordestructive interference between reflections from the various layers.Interference filters are also referred to in the art as dichroicfilters, or dielectric filters. The term can include an absorptivefilter which prevents passage of radiation having a selective wavelengthor wavelength range by absorption. Absorptive filters include, forexample, colored glass or liquid.

A filter used in the invention can have one or more particular filtertransmission characteristics including, for example, bandpass, shortpass and long pass. A band pass filter selectively passes radiation in awavelength range defined by a center wavelength of maximum radiationtransmission (Tmax) and a bandwidth and blocks passage of radiationoutside of this range. Tmax defines the percentage of radiationtransmitted at the center wavelength. The bandwidth is typicallydescribed as the full width at half maximum (FWHM) which is the range ofwavelengths passed by the filter at a transmission value that is half ofTmax. A band pass filter useful in the invention can have a FWHM of 10nanometers (nm), 20 nm, 30 nm, 40 nm or 50 nm. A long pass filterselectively passes higher wavelength radiation as defined by a Tmax anda cut on wavelength. The cut on wavelength is the wavelength at whichradiation transmission is half of Tmax; as wavelength increases abovethe cut on wavelength, transmission percentage increases and aswavelength decreases below the cut on wavelength transmission percentagedecreases. A short pass filter selectively passes lower wavelengthradiation as defined by a Tmax and a cut off wavelength. The cut offwavelength is the wavelength at which radiation transmission is half ofTmax; as wavelength increases above the cut off wavelength, transmissionpercentage decreases and as wavelength decreases below the cut offwavelength transmission percentage increases. A filter of the inventioncan have a Tmax of 50-100%, 60-90% or 70-80%.

As used herein, the term “microarray” refers to a population ofdifferent probe molecules that are attached to one or more substratessuch that the different probe molecules can be differentiated from eachother according to relative location. An array can include differentprobe molecules, or populations of the probe molecules, that are eachlocated at a different addressable location on a substrate.Alternatively, a microarray can include separate substrates each bearinga different probe molecule, or population of the probe molecules, thatcan be identified according to the locations of the substrates on asurface to which the substrates are attached or according to thelocations of the substrates in a liquid. Exemplary arrays in whichseparate substrates are located on a surface include, withoutlimitation, a Sentrix® Array or Sentrix® BeadChip Array available fromIllumina®, Inc. (San Diego, Calif.) or others including beads in wellssuch as those described in U.S. Pat. Nos. 6,266,459, 6,355,431,6,770,441, and 6,859,570; and PCT Publication No. WO 00/63437, each ofwhich is hereby incorporated by reference. Other arrays having particleson a surface include those set forth in US 2005/0227252; WO 05/033681;and WO 04/024328.

Further examples of commercially available microarrays that can be usedin the invention include, for example, an Affymetrix® GeneChip®microarray or other microarray synthesized in accordance with techniquessometimes referred to as VLSIPS™ (Very Large Scale Immobilized PolymerSynthesis) technologies as described, for example, in U.S. Pat. Nos.5,324,633; 5,744,305; 5,451,683; 5,482,867; 5,491,074; 5,624,711;5,795,716; 5,831,070; 5,856,101; 5,858,659; 5,874,219; 5,968,740;5,974,164; 5,981,185; 5,981,956; 6,025,601; 6,033,860; 6,090,555;6,136,269; 6,022,963; 6,083,697; 6,291,183; 6,309,831; 6,416,949;6,428,752 and 6,482,591, each of which is hereby incorporated byreference. A spotted microarray can also be used in a method of theinvention. An exemplary spotted microarray is a CodeLink™ Arrayavailable from Amersham Biosciences. Another microarray that is usefulin the invention is one that is manufactured using inkjet printingmethods such as SurePrint™ Technology available from AgilentTechnologies. Other microarrays that can be used in the inventioninclude, without limitation, those described in Butte, Nature ReviewsDrug Discov. 1:951-60 (2002) or U.S. Pat Nos. 5,429,807; 5,436,327;5,561,071; 5,583,211; 5,658,734; 5,837,858; 5,919,523; 6,287,768;6,287,776; 6,288,220; 6,297,006; 6,291,193; and 6,514,751; and WO93/17126; WO 95/35505, each of which is hereby incorporated byreference.

As used herein, the term “time delay integration” or “TDI” is intendedto mean sequential detection of different portions of a sample bydifferent subsets of elements of a detector array, wherein transfer ofcharge between the subsets of elements proceeds at a rate synchronizedwith and in the same direction as the apparent motion of the samplebeing imaged. For example, TDI can be carried out by scanning a samplesuch that a frame transfer device produces a continuous video image ofthe sample by means of a stack of linear arrays aligned with andsynchronized to the apparent movement of the sample, whereby as theimage moves from one line to the next, the stored charge moves alongwith it. Accumulation of charge can integrate during the entire timerequired for the row of charge to move from one end of the detector tothe serial register (or to the storage area of the device, in the caseof a frame transfer CCD).

As used herein, the term “collection arm” is intended to mean an opticalcomponent or set of optical components positioned to direct radiationfrom a sample region to a detector.

While only certain features of the invention have been illustrated anddescribed herein, many modifications and changes will occur to thoseskilled in the art. It is, therefore, to be understood that the appendedclaims are intended to cover all such modifications and changes as fallwithin the true spirit of the invention.

1. A system for imaging a microarray comprising: a laser light source; asingle mode fiber optic cable coupled to the laser light source fortransmitting laser light in single mode transmission; a line illuminatorfor converting laser light from the source to a line of radiation, theline illuminator including a collimator arranged to receive the laserlight from the source and an aspherical lens for converting collimatedlight from the collimator to the line of radiation; and a focusingdevice for directing the line of radiation onto a plane at the surfaceof a microarray.
 2. The system of claim 1, wherein the line illuminatoris configured to convert laser light from the source to a line ofradiation of substantially uniform intensity over a desired line length.3. The system of claim 1, wherein the collimator and aspherical lens areprealigned within a module to permit installation of the module in thesystem without further alignment within the module during installation.4. The system of claim 1, wherein the focusing device is configured tocreate a uniform line of radiation that is diffraction limited in thenarrow dimension of the line.
 5. The system of claim 1, furthercomprising a laser line filter disposed in the module intermediate thecollimator and the aspherical lens.
 6. The system of claim 1, whereinthe aspherical lens is a Powell lens.
 7. The system of claim 1, whereinthe aspherical lens is a cylindrical lens.
 8. The system of claim 1,wherein individual sites on the microarray are separated by a distancein the range of about 0.1 to 50 micrometers.
 9. The system of claim 8,wherein the system is configured to distinguish the individual sites.10. The system of claim 1, wherein the system is configured to obtain animage of the microarray at a Rayleigh resolution between about 0.2 and10 micrometers.
 11. The system of claim 1, wherein the line illuminatorincludes an integral connector, and the single mode fiber optic cable isterminated with a mating connector for coupling to the line illuminator.12. The system of claim 1, wherein one end of the single mode fiberoptic cable is integrally coupled to the line illuminator.
 13. Thesystem of claim 1, wherein one end of the single mode fiber optic cableis removably coupled to the line illuminator.
 14. The system of claim 1,comprising a second fiber optic cable coupled to the laser light sourceat one end thereof and coupled to the single mode fiber optic cable atan opposite end thereof.
 15. The system of claim 14, wherein the secondfiber optic cable is a single mode fiber optic cable.
 16. The system ofclaim 15, wherein the fiber optic cables are coupled to one another viaan optical connector.
 17. The system of claim 15, wherein the fiberoptic cables are spliced to one another.
 18. The system of claim 1,wherein the single mode fiber optic cable is configured for single modetransmission of laser light with a wavelength of approximately 405 nm,488 nm, 532 nm or 633 nm.
 19. A system for imaging a microarraycomprising: a laser light source; a fiber optic cable coupled to thelaser light source; a line illuminator for converting laser light fromthe source to a line of radiation, the line illuminator including acollimator arranged to receive the laser light from the source and anaspherical lens for converting collimated light from the collimator tothe line of radiation; a second fiber optic cable coupled to the firstsingle mode fiber optic cable at one end thereof and to the lineilluminator at another end thereof, at least one of the fiber opticcables being a single mode fiber optic cable; and a focusing device fordirecting the line of radiation onto a plane at the surface of amicroarray.
 20. A system for imaging a microarray comprising: first andsecond laser light sources, each source configured to output laser lightin a different predetermined frequency band; first and second singlemode fiber optic cables coupled to the first and second laser lightsources, respectively, for transmitting laser light in single modetransmission; and first and second line illuminators coupled to thefirst and second single mode fiber optic cables, respectively, forconverting laser light from the respective source to a line ofradiation, the line illuminators each including a collimator arranged toreceive the laser light from the source and an aspherical lens forconverting collimated light from the collimator to the line ofradiation; and a focusing device for directing the line of radiationonto a plane at the surface of a microarray.
 21. The system of claim 20,further comprising: a combiner for combining the lines of radiation fromthe first and second illuminators; means for confocally irradiating themicroarray with the combined lines of radiation and for returningradiation from the microarray; and a detector for receiving the returnedradiation and for generating signals for use in analysis of themicroarray.
 22. A method for making a system for imaging a microarraycomprising: placing a laser light source, a line illuminator, a focusingdevice and a stage in a configuration wherein: the laser light source iscoupled to the line illuminator via a single mode fiber optic cable, thefiber optic cable configured for transmitting laser light from thesource in single mode transmission, and the line illuminator configuredfor converting laser light from the source to a line of radiation, theline illuminator includes a collimator arranged to receive the laserlight from the source and an aspherical lens for converting collimatedlight from the collimator to the line of radiation; the focusing devicedirects the line of radiation toward a plane; and the stage isconfigured to place a microarray surface at the plane.
 23. A method forimaging a microarray comprising: generating laser light; transmittingthe laser light to a line illuminator via a single mode fiber opticcable, the fiber optic cable configured for transmitting laser lightfrom the source in single mode transmission, and the line illuminatorconfigured for converting laser light from the source to a line ofradiation, the line illuminator including a collimator arranged toreceive the laser light from the source and an aspherical lens forconverting collimated light from the collimator to the line ofradiation; and directing the line of radiation with a focusing device,wherein the line of radiation is directed onto a plane at the surface ofa microarray.
 24. A method for imaging a microarray comprising:generating laser light at first and second wavelengths; transmitting thefirst and second wavelength laser light to respective line illuminatorsvia respective first and second single mode fiber optic cables, eachfiber optic cable configured for transmitting laser light from therespective source in single mode transmission, and each line illuminatorconfigured for converting laser light from the respective source to aline of radiation, each line illuminator including a collimator arrangedto receive the laser light from the source and an aspherical lens forconverting collimated light from the collimator to the line ofradiation; combining the lines of radiation from the line illuminators;directing the combined lines of radiation with a focusing device,wherein the line of radiation is directed onto a plane at the surface ofa microarray, thereby confocally irradiating the microarray with thecombined lines of radiation; and directing radiation returned from themicroarray to a detector configured to generate signals for analysis ofthe microarray.